Native-PAGE

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Native PAGE Principle:

Native PAGE uses the same discontinuous chloride and glycine ion fronts as SDS-PAGE to form moving boundaries that stack and then separate polypeptides by charge to mass ratio. Proteins are prepared in a non-reducing non-denaturing sample buffer, which maintains the proteins’ secondary structure and native charge density. Therefore you can easily see multiple bands from the camshot of your native PAGE gel if your target protein has polymerized forms in your sample. In native PAGE electrophoresis most proteins have an acidic or slightly basic pl (isoelectric point) (~3–8) and migrate towards the negative polar. If your protein's pl is larger than 8,9, for example, you should probably reverse the anode and run the native PAGE gel.

Learn more about Native-PAGE:

Native-PAGE Types

Native-PAGE Tips

Native-PAGE Staining Methods

Native-PAGE Gel Storage

Buffers:

  • For the electrophoresis system, a bio-rad system is recommended.
  • For a 5ml native PAGE stacking gel

 

0.375 M Tris-HCl pH=8.8

4.275ml

Acrylamide/Bis-acrylamide
(30%/0.8% w/v)

0.67ml

*10% (w/v) ammonium persulfate  (AP)

0.05ml

*TEMED

5μl

*: Added right before each use.

  • For a 10ml native PAGE separating gel:

Acylamide percentage

6%

8%

10%

12%

15%

Acrylamide/Bis-acrylamide
(30%/0.8% w/v)

2ml

2.6ml

3.4ml

4ml

5ml

0.375M Tris-HCl(pH=8.8)

7.89ml

7.29ml

6.49ml

5.89ml

4.89ml

*10% (w/v) ammonium persulfate  (AP)

100μl

100μl

100μl

100μl

100μl

*TEMED

10μl

10μl

10μl

10μl

10μl

*: Added right before each use.

  • Sample buffer (2x):

62.5 mM Tris-HCl, pH 6.8

25% glycerol Glycerol

1% Bromophenol Blue

  • Running Buffer:

25 mM Tris

192 mM glycine

Note: running buffer should be~ pH 8.3. Do not adjust the pH.

 

Gel running protocol:

1. Prepare appropriate amount of separating gel in a small beaker, then add specific vol. of AP and TEMED and gently swirl the beacker to ensure a sufficient mixing. Pipet the gel solution into the gap between the glass plates of gel casting (Don't fully fill). Fill the rest space with water (isopropanol alternatively). Allow 20-30min for a complete gelation.

2. You can prepare the stacking gel solution while the separating gel is gelating. Prepare appropriate amount of stacking gel in a beacker and mix with 10% AP and 1% TEMED. Pour out the water in the first step and pipet the stacking gel solution into the gap and insert the comb. Allow 20-30min to let it gelate.

3. Mix your sample with sample buffer. Do not heat your sample!

4. Load the sample mixture and set an appropriate voltage to run the electrophoresis.

Note: It's better to put the system on ice and not set a relative high Volt in case the proteins degrade.

5. Stain as you would a standard Coomassie-blue protocol or proceed to a immuno-blotting procedure (western-blot).